Work in Project 2 will explore exciting recent developments in the study of cross-reactive immunity to influenza viruses. The goal of this project is to determine the molecular and structural basis for broadly cross-reactive human mAb responses to influenza HAs; As the epitope sequences and structures recognized by broadly cross-reactive Abs are defined, we will be better able to rationally design broadly protective immunogens. The work seeks to investigate the repertoire of human Abs recognizing three principal conserved regions of the HA molecule; a canonical stalk domain epitope, the long alpha-helix stalk domain, and the globular head domain. The hypothesis is that, unexpectedly, there are at least three immunogenic domains in influenza HA that retain highly conserved structural features and that most adult healthy subjects do possess circulating cross-reactive B cell clones to these conserved sites on HA. These domains are complex, however, and relatively inaccessible-for conventional Ab structures, we have developed robust technologies for isolating human mAbs that reveal the relatively frequent nature of cross-reactive B cells, and the genetic and structural basis for broad cross-reactivity of the secreted Abs for influenza viruses of diverse subtypes. We hypothesize that such cross-reactivity is driven by unusual features in the Ab repertoire, especially an exceptionally high level of somatic point mutations and somatic insertions. This highly interactive project will engage in collaborative work with Project 1 to determine mAb-HA co-crystal structures. Project 4 to study the biology of viral escape mutant viruses, Project 5 to examine the effect of mutants on DCs, and will use the Glycan Array core to study glycosyiation in escape sites and the Animal Core to determine in vivo potency of antibodies. Studies of such Abs and the sites recognized by them will inform the rational design of universal influenza vaccines based on the underlying principles of heterosubtypic immunity. The stalk domain is of interest because highly cross-reactive Ab have been identified that recognized a conserved region in this area. However, the rare Abs identified to date do not possess both group 1 and 2 specificities. In preliminary data we show that it is possible to isolate such Abs. Using these unusual reagents, we will define the basis for the group determinants or conserved elements: by mapping the interaction of large panels of new Abs to the: stalk region. We also will seek to isolate novel human Abs from human subjects that recognize the stalks of both major HA antigenic groups. The head domain is better studied, however there are only rare Abs that exhibit cross-reactivity for antigenically distinct HAs. By large-scale screening of B cells from subjects immunized with seasonal and experimental vaccines, we have identified cross-reactive Abs that recognize the head domain of HAs of multiple subtypes of influenza. By epitope mapping using biochemical arid biologic studies, coupled with studies to determine co-crystal structures, we will determine the structural basis for cross reactivity.